Knight:Colony PCR protocol

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Solutions/reagents:

• PCR supermix
• VF2
  
  (40 µM)
• VR
  
  (40 µM)
• colony template

Equipment:

• Thermocycler
• Electrophoretic unit
• Reaction tubes

Steps:

1. Reaction Mixture
   Use the following table as a checklist for preparing the reaction in reaction tube (1):
   
   

   	PCR supermix	VF2	VR	colony template
   Colony PCR	9 µl	0.25 µl	0.25 µl	0.5 µl

2. PCR conditions
   Program a standard thermocycler to run the reaction using the following parameters:
   Initial denaturation
   
   • Denature: 95°C, 15 mins
   Thermocycling
   
   • No. of cycles: 39
   • Denature: 94°C, 30 secs
   • Anneal: 56°C, 30 secs
   • Elongate: 68°C, 60 secs
   Elongation time : 1 min per kb of expected product. I typically round up for this step. i.e. For a 3.6kb construct, I used a 4 min elongation time. It seems to help to be a bit generous with the elongation time.
   Termination
   • Elongate: 60°C, 20 mins
   • Hold: 4°C, until removed from machine
3. Perform agarose gel electrophoresis of appropriate quantity of PCR products mixed with ethidium bromide and visualize with UV transilluminator to confirm the presence of required product.
   

TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 1 hr, 55 mins